In the past two years, elevated HEF1 expression have been shown to induce glioblastoma invasion and lung cancer and melanoma metastasis in both animal models and in humans: our preliminary studies have now suggested an equally important role for HEF1 in breast cancer progression. Our studies of HEF1 since we first described this protein in 1996 have elucidated a complex function. HEF1 acts to assemble signaling complexes at focal adhesions in interphase cells, at the centrosome in pre-mitotic cells, and at the cilium in cells emerging from quiescence. Taken in sum, these data suggest that deregulation of HEF1 may represent a point of cellular vulnerability based on coordinate dysregulation of these discrete signaling complexes. If so, restriction of HEF1 action may offer a useful therapeutic approach. The goal of this proposal is to better understand the mechanisms by which the HEF1 scaffolding protein controls both cell cycle progression and cell attachment signaling. Our work in past cycles of R01 CA63366 defined interactions of HEF1 with the oncoproteins Src and FAK as critical for HEF1 action in promoting cell migration and invasion. Subsequently, we defined HEF1 interactions with Aurora-A, Ajuba, and HDAC6 as essential for regulation of mitotic progression and ciliary disassembly. These two sets of signaling relationships have been considered in isolation. However, accumulating evidence suggests roles for Src in mitosis, and for Ajuba and HDAC6 in interphase cells. We propose that HEF1 is a core component of a signaling module that functions both in mitosis and interphase cells, and timed migration of HEF1 and its partners between intracellular compartments allows coordinated regulation of cell division and cell migration/invasion. We propose deregulated HEF1 expression most commonly marks later rather than earlier stages of cancer development because prior lesions inactivating mitotic checkpoints allow cells to tolerate HEF1 scaffolding defects. This proposal has 3 aims. In Aim 1, we will assess the factors regulating HEF1 movement from focal adhesions to centrosomes, allowing HEF1 activation of AurA at mitotic entry. We will determine whether Src and FAK contribute directly to this process. In Aim 2, we will define the role of Aurora A, Ajuba, and HDAC6 in allowing HEF1 to return to focal adhesions at cytokinesis, and the importance of interactions between HEF1, Ajuba, and HDAC6 in interphase for HEF1-dependent cell migration and invasion. In Aim 3, we will use murine knockout and transgenic models to study HEF1-dependent breast cancer development, and to analyze the relationship between deregulated HEF1 expression, genomic instability, and metastasis. PUBLIC HEALTH RELEVANCE: Changes in HEF1 protein expression drive metastasis in more than 30% of human melanomas, and also have been linked to metastatic behavior in glioblastomas, lung cancers, and breast cancers. The proposed work builds from our prior studies analyzing HEF1 interaction with other proto-oncogenic proteins, will elucidate the action of HEF1 in cancer progression, and may reveal points of cancer cell vulnerability that can be therapeutically exploited.